This invention relates to a method for improving the growth and regeneration potential of embryogenic cell and tissue cultures of coniferous plants retrieved from cryopreservation. In particular, this invention relates to the use of abscisic acid in the post-cryopreservation recovery medium to improve both the growth and somatic embryo production of embryogenic cell and tissue cultures of conifers, thereby enabling more rapid proliferation of the embryogenic cultures and a subsequent increase in the yield of somatic embryos. This method is well-suited for employment with a number of biotechnological uses of embryogenic cultures of coniferous plants retrieved from cryopreservation, including use with embryogenic cultures of coniferous plants and with genetically transformed embryogenic cultures of coniferous plants for producing clonal planting stock useful for reforestation.
Cryopreservation is the storage of living cells at ultra-low (cryogenic) temperatures, usually in liquid nitrogen (xe2x88x92196xc2x0 C.) or in its vapor phase (about xe2x88x92150xc2x0 C.). Cryopreservation is the preferred method for long-term storage and xe2x80x9cbankingxe2x80x9d of valuable in vitro biological material used in or derived from biotechnology. At cryogenic temperatures the biological activity of the cells and tissues is halted. The cells and tissues remain viable throughout the cryopreservation process due to the application of various cryoprotective procedures (Benson et al. 1998).
There are several methods of freezing used in cryopreservation of biological materials such as living cells or tissues. The basic method is to rapidly cool the biological material or to directly plunge the material into liquid nitrogen. However, this method only works on tissues which remain viable at low moisture content levels. For example, many temperate zone seeds (such as pine seeds which have been dried to below about 15% water content) can be successfully cryopreserved using this method.
A different cryopreservation method is frequently used where the biological material has a relatively high moisture content or where the material may not tolerate dehydration to a lower moisture level. This method involves first treating the biological material with a cryoprotective chemical or combination of cryoprotective chemicals. The treated material is subsequently cooled to about xe2x88x9240xc2x0 C., then rapidly cooled (e.g., directly plunged into liquid nitrogen) to cryogenic temperatures. This method is the one most frequently employed for cryopreservation of in vitro derived cells and tissues.
Another well-known cryoprotective method is xe2x80x9cvitrification.xe2x80x9d This method involves the treatment of biological materials with high levels of cryoprotective chemicals in combination with a rapid cooling of the treated materials to cryogenic temperature.
Regardless of the cryogenic freezing method employed, recovery of viable cells after cryopreservation is dependent upon both pre-cryopreservation and post-cryopreservation treatments. In vitro manipulation of the plant tissues or cells in the second and third cryogenic freezing methods noted above are essential to most pre-cryopreservation and post-cryopreservation recovery protocols (Benson et al. 1998). Pre-cryopreservation treatments commonly include the application of a cryoprotective chemical such as glycerol or dimethyl sulfoxide (DMSO). Also, the osmotic potential of the in vitro culture medium is often decreased in the pre-cryopreservation treatment of tissues or cells via the addition of sugars or sugar alcohols such as sucrose, sorbitol, and the like. Post-cryopreservation treatments commonly include the dilution of the cryoprotective chemicals and the osmoticants in the culture medium. Such traditional pre-cryopreservation and post-cryopreservation procedures are commonly practiced and are familiar to those skilled in the art of cryopreservation of plant tissues and cells.
Culture media in which the tissues or cells are grown and proliferated during both the pre-cryopreservation and post-cryopreservation phases typically contain six groups of ingredients: inorganic nutrients, vitamins, organic supplements, a carbon source (i.e., sugars), phytohormones (e.g., auxins or auxins and cytokinins), and a gelling agent for semi-solid or gelled medium. Thus, a commonly used pre-cryopreservation medium for embryogenic cultures would include a standard culture medium (e.g., a medium containing inorganic nutrients, vitamins, organic supplements, and sucrose like that taught by Murashige and Skoog (1962) or a modification thereof) coupled with an auxin, possibly a cytokinin, sorbitol, and DMSO. A typical post-cryopreservation medium for embryogenic cultures would include a standard culture medium, an auxin and possibly a cytokinin, but would be devoid of the cryoprotective chemicals and additional osmotic agents. Frequently, the tissues and cells are initially cultured during post-cryopreservation for a very brief period (typically, one day) on a temporary recovery medium to allow both the cryoprotective chemicals and the additional osmotic agents used in the pre-cryopreservation medium to diffuse out of the tissue and cells. The tissues or cells are then transferred to the same (fresh) medium, lacking cryoprotective chemicals and osmotic agents, to induce recovery and growth. The actively growing tissues or cells can then be utilized for regeneration of plants, or for other biotechnological and genetic engineering purposes.
A significant problem facing those who work with cells and tissue cultures from trees is how to rapidly recover and multiply by proliferation the embryogenic cultures during the post-cryopreservation phase of the somatic embryogenesis process. Somatic embryogenic cultures are employed in the regeneration of trees for clonal propagation, and in the genetic transformation and subsequent regeneration of transgenic trees. It is well-known that embryogenic cultures in general, and pine embryogenic cultures in particular, decline in regeneration potential as the time in culture increases. It is, therefore, important to decrease the length of time taken to multiply or bulk-up the cultures for use in clonal propagation or genetic transformation. It is also believed that increased time in culture may increase the probability of deleterious genetic changes or mutations that result in unwanted somaclonal variation. Such variations are particularly undesirable in clonal propagation and genetic engineering processes. Thus, a central problem or challenge in somatic embryogenesis systems is the need to keep the time in culture to a minimum, while simultaneously producing large amounts of embryogenic tissue or cells which have the potential to produce large numbers of harvestable somatic embryos. This invention addresses the restraints imposed on such systems due to slow growth and recovery during a specific step of the process, namely, the post-cryopreservation recovery phase.
Propagation by somatic embryogenesis refers to methods whereby embryos are produced in vitro from embryogenic cultures. The embryos are referred to as somatic because they are derived from the somatic (vegetative) tissue, rather than from the sexual process. Vegetative propagation via somatic embryogenesis has the capability to capture all genetic gain of highly desirable genotypes. Furthermore, these methods are readily amenable to automation and mechanization. These qualities endow somatic embryogenesis processes with the potential to produce large numbers of individual clones for reforestation purposes.
It was not until 1985 that somatic embryogenesis was discovered in conifers (Hakman et al. 1985) and the first viable plantlets were regenerated from conifer somatic embryos (Hakman and von Arnold 1985). Since 1985, conifer tissue culture workers throughout the world have pursued the development of somatic embryogenesis systems capable of regenerating plants. The goal of much of this work is to develop conifer somatic embryogenesis as an efficient propagation system for producing clonal planting stock en masse. Additionally, the embryogenic system interfaces very well with genetic engineering techniques for production of transgenic clonal planting stock of conifers.
The somatic embryogenesis processes utilized with coniferous plants usually involves seven general steps: 1) culture initiation, 2) culture maintenance, 3) embryo development, 4) embryo maturation, 5) embryo germination, 6) conversion, and 7) field planting. An additional step, cryopreservation, has become an integral component in conifer somatic embryogenesis. Cryopreservation is important because it is necessary to determine the genetic potential of plants regenerated from each culture before clonal planting stock can be produced. This determination of the genetic potential is done in a xe2x80x9cclonal field testxe2x80x9d which typically takes four to six years to complete. Therefore, cryopreservation of the embryogenic cultures, which are represented in the clonal field test, has become the preferred way for long-term storage during the completion of the clonal field test. While cryopreservation of embryogenic cultures can be practiced throughout the somatic embryogenesis process, it is commonly practiced during the first three general steps noted above; and preferably practiced with newly initiated and/or maintained embryogenic cultures.
The importance of abscisic acid (ABA) during the development and maturation of zygotic embryos is well known, and ABA has been used routinely to stimulate embryo development in somatic embryogenic systems (von Arnold and Hakman, 1988). For example, U.S. Pat. No. 4,957,866 teaches the use of ABA in the embryo development media. Likewise, in U.S. Pat. Nos. 5,034,326 and 5,036,007 the phytohormone ABA along with activated carbon has been reported to be beneficial in the semi-solid development media for various conifers. U.S. Pat. No. 5,294,549 teaches the incorporation of ABA and gibberellic acid into the embryo development media. U.S. Pat. Nos. 5,187,092, 5,183,757, and 5,236,841 teach the use of ABA in the development step in conifer somatic embryogenesis. In each of these methods ABA is added for the purpose of facilitating embryo development.
U.S. Pat. No. 5,856,191 by Handley (1999) employs ABA in both the initiation and maintenance medium for pine embryogenic cultures prior to cryopreservation. The use of ABA in these earlier phases of the pine somatic embryogenesis process differs from the use of ABA in the post-cryopreservation recovery medium taught by the current invention. That is, while U.S. Pat. No. 5,856,191 claims the cryopreservation of pine embryogenic cultures that have been initiated on culture medium containing ABA, the current invention teaches the use of ABA in the post-cryopreservation phase of culture growth and recovery from cryogenic storage. It has been found that the coupling of the present method with the process taught in U.S. Pat. No. 5,856,191 yields a marked improvement in the growth of embryogenic cultures during the critical phase of recovery from cryogenic storage. Indeed, there are clear advantages to using ABA in both the initiation and maintenance phase prior to cryopreservation (as taught in U.S. Pat. No. 5,856,191), and in subsequently employing ABA in the post-cryopreservation phase of culture growth and recovery from cryogenic storage as taught in the current invention.
Heretofore there has been no evidence that the use of ABA in the phase of recovery of embryogenic cells from cryogenic storage, either in plants in general or with coniferous species, would be beneficial. In fact, although it is well known that ABA is important in the development of embryos both in vivo and in vitro, the ability of ABA to stimulate proliferation of embryogenic tissue growth in material emerging from cryogenic storage was unexpected.
ABA has been added to a pre-cryopreservation medium to successfully cryopreserve wheat zygotic embryos (Kendall et al. 1993). However, the effect of ABA to improve the recovery and growth during the post-cryopreservation step was not examined or suggested in this study. The wheat embryos resumed growth when cultured during the post-cryopreservation step on an MS-based medium containing an auxin, but devoid of ABA. While the treatment of currant meristems and callus during the pre-cryopreservation phase with an ABA-responsive protein was shown to improve recovery after a vitrification method of freezing (Luo and Reed 1997), the study did not suggest the use of, or examine the effect of, adding ABA to the post-cryogenic medium to improve recovery and growth. Indeed, we are not aware of any work that teaches the use ABA to improve the growth of embryogenic cultures of plants, including conifers, during the post-cryopreservation step.
Therefore, an object of the present invention is to provide a method for improving the recovery and growth of embryogenic cultures of coniferous plants recovered from long-term or short-term cryopreservation.
A further object of the present invention is to improve the yield of somatic embryos obtained from the proliferating embryogenic cultures of coniferous plants recovered from long-term or short-term cryopreservation.
Another object of the present invention is to decrease the time required to multiply and proliferate the embryogenic cultures of coniferous plants retrieved from cryopreservation.
A further object of the present invention is to provide an improved method for the recovery of embryogenic cultures of the genus Pinus and Pinus interspecies hybrids from cryopreservation so that these cultures can be used to regenerate clonal planting stock via somatic embryogenesis.
Another object of the present invention is to provide an improved method for the recovery of embryogenic cultures of the genus Pinus and Pinus interspecies hybrids which have been genetically engineered to contain at least one transgene from cryopreservation so that these cultures can be used to regenerate transgenic clonal planting stock via somatic embryogenesis.
The above objectives are achieved by the use of an improved method for recovering embryogenic cultures of coniferous plants from cryopreservation. This method enables the practitioner to increase the growth of embryogenic cell lines retrieved from cryogenic storage, and thus more quickly have large amounts of embryogenic tissue available for subsequent use. In addition, this method enables the practitioner to increase the yield of somatic embryos harvested from the embryogenic cultures recovered from cryopreservation. This was accomplished via the addition of abscisic acid to the post-cryopreservation recovery medium on which the thawed embryogenic culture was placed for growth. The abscisic acid is utilized in combination with standard (traditional) phytohormones employed during the post-cryopreservation recovery phase.
This method is well-suited for use with coniferous embryogenic cultures for producing clonal planting stock useful for reforestation. Likewise, the present method can be employed in conjunction with genetically transformed coniferous embryogenic cultures for producing transgenic clonal planting stock useful for reforestation.
The present invention is a method for recovering living embryogenic cultures of coniferous plants which have been subjected to cryopreservation which comprises: 1) thawing the embryogenic culture, and 2) culturing the thawed embryogenic culture on post-cryopreservation recovery medium containing a sufficient amount of nutrients, a suitable level of gelling agent, abscisic acid, and a sufficient amount of additional phytohormone for a sufficient period of time, under suitable environmental conditions, to recover (i.e., proliferate and multiply) the embryogenic culture.
Where warranted, an additional step may be employed to allow undesired cryoprotective chemicals and/or osmotic agents to diffuse out of the embryogenic cultures. This method for recovering living embryogenic cultures of coniferous plants which have been subjected to cryopreservation comprises: 1) thawing the embryogenic culture, 2) culturing the thawed embryogenic culture on a temporary recovery medium containing a sufficient amount of nutrients, suitable level of gelling agent, and a sufficient amount of phytohormone for a period of time sufficient to lower the concentration of cryoprotective chemicals and osmotic agents contained in the embryogenic culture; and 3) further culturing the thawed embryogenic culture on post-cryopreservation recovery medium containing a sufficient amount of nutrients, a suitable level of gelling agent, abscisic acid and a sufficient amount of additional phytohormone for a sufficient period of time, under suitable environmental conditions, to recover (i.e., proliferate and multiply) the embryogenic culture.
The current method of using ABA in the post-cryopreservation medium can be employed in methods used to regenerate conifers via somatic embryogenesis. Methods to regenerate Pinus and Pinus interspecies hybrids via somatic embryogenesis are taught in U.S. Pat. Nos. 5,491,090; 5,506,136; 5,731,191; 5,731,204; and 5,856,191 (which are hereby incorporated by reference). Likewise, the current method can be employed to regenerate via somatic embryogenesis conifers which have been genetically transformed to contain at least one transgene. Embryogenic cultures recovered via the present method also exhibit improved production and yields of somatic embryos.
The present invention lies in the incorporation of abscisic acid (ABA) into the post-cryopreservation recovery medium of conifer embryogenic cell cultures. A suitable level of ABA for use in this invention is from about 1.0 to about 100.0 milligrams per liter of medium (mg/l) of medium. The preferred ABA level is about 5.0 to about 50.0 mg/l.
While most cell lines exhibit improved growth rates when grown on post-cryopreservation recovery medium containing ABA than when grown on medium which lacks ABA, the degree of improved growth of such embryogenic cultures depends in part upon the cell line employed. It may be possible in the future to better determine the morphological and/or biochemical differences among cell lines which quantify the degree of improved growth-response to ABA in the post-cryopreservation recovery medium.
The present method is suitable for use with embryogenic cultures of coniferous plants. It is preferred to employ the present method with conifers of the genus Pinus and Pinus interspecies hybrids. This method is generally applicable to somatic tissue obtained from the Pinus species including, but not limited to, the following: Pinus taeda (loblolly pine), P. elliottii (slash pine), P. palustris (longleaf pine), P. serotina (pond pine), P. echinata (shortleaf pine), P. clausa (sand pine), P. glabra (spruce pine), P. rigida (pitch pine), P. echinata (shortleaf pine), P. nigra (Austrian pine), P. resinosa (red pine), P. sylvestris (Scotch pine), P. banksiana (jack pine), P. virginiana (Virginia pine), P. radiata (Monterey pine), P. contorta (shore pine), P. contorta latifolia (lodgepole pine), P. ponderosa (ponderosa pine), P. leiophylla (Chihuahua pine), P. jeffreyi (Jeffrey pine), and P. engelmannii (Apache pine), P. strobus (eastern white pine), P. monticola (western white pine), and P. lambertiana (sugar pine), P. albicaulis (whitebark pine), P. flexilis (limber pine), P. strobiformis (southwestern white pine), P. caribaea (Caribbean pine), P. patula (Mexican weeping pine), P. tecumumanii (Tecun Uman pine), P. maximinoi, P. oocarpa (Ocote Pine) and P. chiapensis (Mexican White pine). In addition, the current invention is specifically applicable to interspecies hybrids of the above mentioned pines including Pinus rigidaxc3x97P. taeda, P. serotinaxc3x97P. taeda, and reciprocal crosses.
It is further preferred to utilize the present method with Southern yellow pines, Pinus rigida, and hybrids thereof. Those skilled in the art recognize that several species of pine indigenous to the Southeastern United States are closely related and hybridize naturally. Taxonomically this group of pines is referred to as xe2x80x9cSouthern yellow pinesxe2x80x9d and includes Pinus taeda, P. serotina, P. palustris, and P. elliottii (Preston 1989). In addition to the taxonomically similarity of the above Southern yellow pine species, these species have also responded similarly in studies on somatic embryogenesis attempts.
It is preferred that the additional phytohormone employed in the post-cryopreservation recovery medium be a member selected from the group consisting of auxins, cytokinins, and combinations thereof. Auxins suitable for use in the present invention include 2,4-D (2,4-dichlorophenoxy acetic acid), NAA (xcex1-Naphthaleneacetic acid), and the like. It is preferred to incorporate a level of about 0.1 to 5.0 mg/l of auxin in the post-cryopreservation recovery medium. Cytokinins suitable for use in the present invention include, but are not limited to, the following: BAP (N6-benzylamino-purine), kinetin (6-Furfurylaminopurine), zeatin (6-[4-hydroxy-3-methylbut-2-enylamino]purine), and combinations thereof. It is preferred to incorporate about 0.1 to 1.0 mg/l of cytokinin in the post-cryopreservation recovery medium. The above-noted levels and types of phytohormones (as well as the phytohormone ABA) are also suitable for use in the temporary recovery medium.
In addition to phytohormones, both the post-cryopreservation and temporary recovery media also require sufficient amounts of nutrients such as inorganic nutrients, vitamins, organic supplements, a carbon source (i.e., sugars), and the like to allow the thawed culture to recover from the cryogenic freezing process and cryopreservation. It is preferred to employ in the media a sugar selected from the group consisting of glucose, maltose, sucrose, and combinations thereof. The preferred amount of sugar is in the range of about 10.0 to 40.0 g/l. However, the present method is not limited to any single culture nutrient medium formulation. It should be understood that any nutrient media commonly used in conifer somatic embryogenesis will be suitable for use with this invention.
Additionally, both the post-cryopreservation recovery medium and the temporary recovery medium require a suitable level of gelling agent. It is preferred to incorporate a level of gelling agent selected from the group consisting of 6.0 to 9.0 g/l of agar, 1.75 to 4.0 g/l of gellan gum, 6.0 to 8.0 g/l of agarose, 3.5 to 5.0 g/l of AGARGEL, and combinations thereof into the recovery media.
The procedures for thawing cryopreserved embryogenic cultures are well-known to skilled artisans. It is preferred that the cryopreserved embryogenic cultures be thawed until ice crystals are no longer present in the cultures. It is further preferred that cryopreserved embryogenic cultures be thawed rapidly by placing the contained cultures in a warm (about 44xc2x0 C.) water bath until the ice crystals have melted.
The preferred period of time for culturing the thawed embryogenic cultures on the post-cryopreservation medium is from about 1 to about 10 weeks. It is further preferred that the thawed embryogenic cultures be subcultured (i.e., transferred to the same, fresh, post-cryopreservation medium) at intervals of from about 1 day to about 3 weeks.
Where a temporary recovery medium is employed, it is preferred to culture the thawed embryogenic culture on the temporary recovery medium for a period of time sufficient to allow the concentration of cryoprotective chemicals and osmotic agents present in the embryogenic culture to be lowered via diffusion into the temporary recovery medium. The period of time commonly employed for such diffusion is from about 1 to about 72 hours; with the preferred period of time being from about 12 to about 48 hours.
A number of terms are known to have differing meanings when used in the literature. The following definitions are believed to be the ones most generally used in the field of plant biotechnology and are consistent with the usage of the terms in the present specification.
A xe2x80x9ccell linexe2x80x9d is a culture that arises from an individual explant.
xe2x80x9cClonexe2x80x9d when used in the context of plant propagation refers to a collection of individuals having the same genetic makeup.
xe2x80x9cConversionxe2x80x9d refers to the acclimatization process that in vitro derived germinating somatic embryos undergo in order to survive under ex vitro (nonaxenic) conditions, and subsequent continued growth under ex vitro conditions.
An xe2x80x9cembryogenic culturexe2x80x9d is a plant cell or tissue culture capable of forming somatic embryos and regenerating plants via somatic embryogenesis.
xe2x80x9cEmbryogenic tissuexe2x80x9d, in conifers, is a mass of tissue and cells comprised of very early stage somatic embryos and suspensor-like cells embedded in a mucilaginous matrix. This has also been referred to as xe2x80x9cembryogenic suspensor massesxe2x80x9d by some researchers and is also called xe2x80x9cembryogenic callusxe2x80x9d in some of the conifer somatic embryogenesis literature; but most researchers believe it is not a true callus.
An xe2x80x9cexplantxe2x80x9d is the organ, tissue, or cells derived from a plant and cultured in vitro for the purpose of starting a plant cell or tissue culture.
xe2x80x9cField plantingxe2x80x9d is the establishment of laboratory, greenhouse, nursery, or similarly grown planting stock under field conditions.
xe2x80x9cFresh weightxe2x80x9d is the weight in grams of a sample of the fully-hydrated embryogenic culture.
xe2x80x9cGenotypexe2x80x9d is the genetic constitution of an organism; the sum total of the genetic information contained in the DNA of an organism.
xe2x80x9cGerminationxe2x80x9d is the emergence of the radicle or root from the embryo.
xe2x80x9cInitiationxe2x80x9d is the initial cellular proliferation or morphogenic development that eventually results in the establishment of a culture from an explant.
xe2x80x9cMegagametophytexe2x80x9d is haploid nutritive tissue of the conifer seed, of maternal origin, within which the conifer zygotic embryos develop.
xe2x80x9cNutrientsxe2x80x9d are the inorganic chemicals (e.g., nitrogen), vitamins, organic supplements, carbon sources, and the like which are necessary for the nourishment of the cultures (resulting in their growth differentiation and/or regeneration).
xe2x80x9cPhytohormonesxe2x80x9d are chemical substances, either naturally occurring or synthesized compounds, which affect the growth, differentiation and development of plant cells, tissues and organs. Phytohormones are frequently applied exogenously to in vitro plant cells, tissues, and organs to achieve desired effects on growth and regeneration.
xe2x80x9cRegenerationxe2x80x9d, in plant tissue culture, is a morphogenic response to a stimuli that results in the production of organs, embryos, or whole plants.
xe2x80x9cSomatic embryogenesisxe2x80x9d is the process of initiation and development of embryos in vitro from somatic cells and tissues.
A xe2x80x9csomatic embryoxe2x80x9d is an embryo formed in vitro from vegetative (somatic) cells by mitotic division of cells. Early stage somatic embryos are morphologically similar to immature zygotic embryos; a region of small embryonal cells subtended by elongated suspensor cells. The embryonal cells develop into the mature somatic embryo.
A xe2x80x9csuspension culturexe2x80x9d is a culture composed of cells and early stage somatic embryos suspended in a liquid medium, usually agitated on a gyratory shaker. An embryogenic suspension culture in conifers is usually composed of early stage somatic embryos with well-formed suspensor cells and dense cytoplasmic head cells that float freely in the liquid medium.
The following examples are provided to further illustrate the present invention and are not to be construed as limiting the invention in any manner.